Biology Reference
In-Depth Information
12.1.1.2
Pull-down assays
To determine the binding of Gs
a
to tubulin, we use pull-down assays with purified
6His-Gs
a
and purified tubulin. 6His-Gs
a
(described above) is loaded with GDP
b
S
(GDP-form) or GTP
g
S (GTP form) and purged of free nucleotide by concentration/
dialysis incubated with tubulin and incubated with a one- or threefold molar ratio of
PC-tubulin/G
a
for 1 h at room temperature. Complexes are then incubated with 50
m
l
of Ni
2
รพ
-nitrilotriacetic acid-agarose beads for 2.5 h at 4
C. The samples are centri-
fuged at 13,000
g
for 15 min and washed with 50 mMTris buffer. These beads bind
6-His, thus binding Gs
a
and any proteins with which it complexes. The samples are
resuspended in 1
SDS sample buffer and separated on 12% SDS-polyacrylamide
gels. The gels are stained with Coomassie Blue before drying. The data show a strong
preference for tubulin binding to the GTP form (Q227L) of Gs
a
(
Fig. 12.2
A).
12.1.1.3
Binding analysis by SPR
The real-time interaction between Gs
a
and tubulin is measured using surface plas-
mon resonance (SPR). The information achieved from this technique can be used to
determine the kinetics of bi-molecular interactions (
Hahnefeld, Drewianka, &
Herberg, 2004; Jason-Moller, Murphy, & Bruno, 2006
).
12.1.1.3.1
Tubulin immobilization on SPR sensor chip
Tubulin is immobilized in HBS-P buffer pH 7.4 at a flow rate of 10
m
l/min on a car-
boxymethyl dextran-coated sensor chip (CM5) using amine coupling according to
the manufacturer's instructions.
N
-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC) are used to link the free
amino acids on tubulin to the sensor chip. Flow cells are injected with 50% NHS/
EDC at a flow rate of 10
m
l/min followed by 100
m
lof1
m
M tubulin in PEM and
1 M ethanolamine (pH 8.5) to prevent further coupling. This resulted in 200-
1000 RU (response unit) of tubulin bound to the sensor chip (1 RU
1 pg/mm
2
pro-
tein). A reference flow cell is injected with PEM instead of tubulin. All reactions are
performed at 25
C. All buffers are freshly prepared using milli-Qwater, filtered, and
degassed before use.
12.1.1.3.2
Detecting the binding between Gsa and tubulin using SPR
Different concentrations of Gs
a
Q227L
and Gs
a
WT (60, 100, and 200 nM) are prepared
freshly in HBS-P buffer and injected onto the flow cell at a flow rate of 30
m
l/min. The
real-time interaction between tubulin and Gs
a
is then monitored as RU.
Figure 12.2
B
shows sensorgrams of real-time interaction between injected Gs
a
Q227L
or Gs
a
WT and
immobilized tubulin. The sensorgram from buffer blank injections and the reference
flow cell (without ligand) are subtracted in order to correct for the buffer shifts created
during injection and the nonspecific binding, respectively. The final curves are then
fitted to a 1:1 Langmuir association model with drifting baseline to obtain the binding
kinetics. Gs
a
Q227L
interacts with tubulin in a concentration-dependent manner. The
affinity of Gs
a
Q227L
binding to tubulin is measured as 100 nM. After each injection,
the surface is regenerated by injection of 1 MNaCl, 0.5% triton X-100 in HBS-P buffer
to removed bound protein. Gs
a
are also injected onto the reference flow cell (no
